EP2660316A1 - Lignée cellulaire aviaire et son utilisation dans la production de protéines - Google Patents

Lignée cellulaire aviaire et son utilisation dans la production de protéines Download PDF

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EP2660316A1
EP2660316A1 EP20120166489 EP12166489A EP2660316A1 EP 2660316 A1 EP2660316 A1 EP 2660316A1 EP 20120166489 EP20120166489 EP 20120166489 EP 12166489 A EP12166489 A EP 12166489A EP 2660316 A1 EP2660316 A1 EP 2660316A1
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Prior art keywords
cell line
cells
protein
avian
process according
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German (de)
English (en)
Inventor
Manfred Wirth
Lijing Sun
Zhiguo Su
Roland Riebe
Volker JÄGER
Nadine Konisch
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National Key Laboratory of Biochemical Engineering
Friedrich-Loeffler-Institut
Helmholtz Zentrum fuer Infektionsforschung HZI GmbH
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National Key Laboratory of Biochemical Engineering
Friedrich-Loeffler-Institut
Helmholtz Zentrum fuer Infektionsforschung HZI GmbH
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Application filed by National Key Laboratory of Biochemical Engineering, Friedrich-Loeffler-Institut, Helmholtz Zentrum fuer Infektionsforschung HZI GmbH filed Critical National Key Laboratory of Biochemical Engineering
Priority to EP20120166489 priority Critical patent/EP2660316A1/fr
Priority to PCT/EP2013/059117 priority patent/WO2013164389A1/fr
Priority to US14/396,336 priority patent/US20150104830A1/en
Priority to EP13723038.9A priority patent/EP2844740A1/fr
Publication of EP2660316A1 publication Critical patent/EP2660316A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16151—Methods of production or purification of viral material
    • C12N2760/16152—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles

Definitions

  • the present invention relates to an avian cell line, suitable for protein production by heterologous expression, e.g. of soluble protein or of viral particles by cultivation of the cells, preferably in suspension in serum-free medium. Further, the invention relates to a process for producing the cell line and to the cell line produced by the process.
  • the cell line Due to the process for generating the cell line of the invention, the cell line has the advantage of being free from mammalian virus, e.g. free from viral nucleic acid sequences originating from virus having specificity for mammals.
  • EP 1483369 B1 , EP 1646715 B1 and EP 1985305 Al describe processes for generating avian cell lines using a specific combination of growth factors and cultivation on a mammalian feeder cell layer, followed by a change of medium involving the stepwise reduction of growth factors, serum components and feeder layer for adaptation of cells to cultivation conditions without feeder cells, and with a reduced content of growth factors and/or of serum.
  • the cell lines carry embryonic stem cell markers, including surface markers SSEA-1, SSEA-3, and show cell renewal.
  • WO 2005/007840 Al describes the production of viral particles using an avian cell line generated by cultivating avian embryonic stem cells on an inactivated feeder cell layer in the presence of growth factors with subsequent stepwise withdrawal of feeder cells and of growth factors during subsequent passages.
  • the final cell lines have telomerase and alkaline phosphatase activities.
  • Avian cells which have come into contact with mammalian cells have the disadvantage of possibly being contaminated with mammalian virus originating from the mammalian cells.
  • WO 2005/042728 Al describes a non-virally transfected cell line which was immortalized by transfecting a first gene disrupting the complex between retinoblastoma protein and E2F transcription factors and by transfecting a second gene affecting protein p53.
  • an alternative avian cell line especially a duck cell line, a method for generating the cell line, and a production process for protein, especially for soluble protein or for viral particles, using the cell line.
  • the alternative cell line is suitable for suspended growth cultivation in a serum-free cell culture medium and is free from endogenous mammalian viral nucleic acid sequences, especially free from an integrated genome of a virus having specificity for mammalian cells.
  • the alternative cell line has a high productivity for both heterologous soluble protein, e.g. for a protein having antigen specificity, especially for a natural or synthetic antibody, and for viral particles, e.g. for proteinaceous viral particles, optionally containing a synthetic nucleic acid construct or a viral genome, e.g. of an attenuated virus.
  • heterologous soluble protein e.g. for a protein having antigen specificity, especially for a natural or synthetic antibody
  • viral particles e.g. for proteinaceous viral particles, optionally containing a synthetic nucleic acid construct or a viral genome, e.g. of an attenuated virus.
  • the invention achieves the objects by the features of the claims, especially by a process for generating an avian cell line, preferably of duck origin, especially originating from Anas platyrhinchos or a domesticated breed thereof, by the following process:
  • the avian embryo is a duck embryo, e.g. of Anas platyrhrinchos or of a domesticated breed originating therefrom.
  • a preferred example for a cell culture medium, especially suitable for cultivating the cells in suspended culture has a composition comprising or consisting of the following ingredients: MEM: L-arginine hydrochloride at 126 mg/L, L-cystine at 24 mg/L, L-glutamine at 292 mg/L, L-histidine hydrochloride-H 2 O at 42 mg/L, L-isoleucine at 52 mg/L, L-leucine at 52 mg/L, L-lysine hydrochloride at 73 mg/L, L-methionine at 15 mg/L, L-phenylalanine at 32 mg/L, L-threonine at 48 mg/L, L-tryptophan at 10 mg/L, L-tyrosine at 36 mg/L, L-valine at 46 mg/L, choline chloride at 1 mg/L, D-calcium pantothenate at 1 mg/L, folic acid at 1 mg/L, niacinamide at 1 mg/L,
  • a preferred example of a cell line according to the invention is deposited under the Budapest treaty under accession number DSM ACC3149, deposited on 12.December 2011 at the Deutsche Stammsammlung für Mikroorganismen und Zellkulturen (DSMZ), Inhoffenstr. 7, 38124 Braunschweig.
  • this cell line is also termed sEFB1.
  • This cell line is also the preferred cell line for the process for production of protein, e.g. of a soluble protein, e.g. a natural or synthetic antibody, or for a viral particle, preferably from a nucleic acid construct that encodes the protein within an expression cassette and is contained in the cell line.
  • an avian cell line can be generated by this process, which cell line both has not been contacted with mammalian cells, e.g. has not been contacted with a feeder cell layer containing mammalian cells, and can be cultivated in suspension in a serum-free medium. It has been found that the cell lines generated by the process are genetically stable and differentiated, showing a fibroblast-like morphology and e.g. lack at least certain embryo-specific markers, e.g. preferably are free from the markers SSEA-1 (stage-specific embryonic antigen 1), SSEA-3 (stage-specific embryonic antigen 3), and EMA-1 (a primordial germ cell marker). The reduction or absence of embryonic markers shows that the cells of the invention are essentially differentiated. In contrast, cells previously described (e.g. EB66) exhibit embryonic markers and therefore retain at least some residual potential for unwanted differentiation and instability.
  • SSEA-1 stage-specific embryonic antigen 1
  • SSEA-3 stage-specific embryonic antigen 3
  • EMA-1 a primordial germ cell marker
  • the cell line of the invention is suitable for protein production at significant concentrations, e.g. by expression of a protein encoded by an expression cassette introduced into the cells, and the cell line is also suitable for production of viral particles at significant titers, including both virus having specificity for mammalian cells, especially for human cells, and virus having specificity for avian cells, especially for duck cells.
  • the process for generating a cell line of the invention shows that the limit to propagation, which can often be found to limit proliferation to approximately 35 to 40 passages can be overcome, even allowing a later reduction, e.g. following at least 35 passages, preferably more than 40 passages, while reducing the content of FBS to about 1 - 2%, followed by an adaptation of the cells to serum-free medium by further passaging in serum-free medium.
  • the cell line of the invention can e.g. be propagated to at least 140 passages in serum-containing medium, preferably in serum-reduced medium, more preferred in serum-free medium without displaying a deterioration of growth characteristics or of protein production, including production of soluble and/or viral particles.
  • the cell lines of the invention combine the properties of being differentiated cells, i.e. with a reduced level of expressed embryonic cell specific marker, preferably without embryonic cell specific markers, especially without embryonic stem cell specific markers, but having the ability for growth, especially in suspended culture, e.g. for more than 50 passages, preferably for more than 70 passages or for more than 140 passages, preferably for unlimited propagation and unlimited proliferation under culture conditions, e.g. in serum-free medium, generally including cultivation in suspension.
  • the cell lines of the invention have a reduced level, e.g.
  • a reduced level of the embryonic cell specific markers is preferably determined by a specific assay, e.g. by an immunological assay for SSEA-1 and SSEA-3 (using anti-SSEA-1 and anti-SSEA -3 antibodies available from DSHB, Developmental Studies Hybridoma Bank, University of Iowa), and by an immunological. assay for EMA-1, respectively.
  • a cell line of the invention is free from the marker if in the specific assay the detected signal shows the absence of marker, e.g. if the detected specific signal is background level.
  • the growth or cultivation of cells in suspended culture or in suspension is the growth of single or agglomerated cell lines of the invention, preferably without attachment to an artificial surface, e.g. without attachment to a carrier.
  • the process for producing protein includes the cultivation of a cell line obtainable by the process for generating the cell lines, preferably in a serum-free cell culture medium, wherein the cells have been genetically manipulated to contain an expression cassette encoding the protein, and/or in the presence of a viral particle which propagates in the cells, optionally in the presence of an auxiliary factor allowing infection of the cells by the viral particle.
  • the expression cassette may contain a nucleic acid sequence encoding the protein to be produced, e.g. a viral particle and/or a viral coat protein or a protein having affinity to an antigen, like e.g. an antibody having a natural or synthetic amino acid sequence.
  • a protein are peptide hormones, cellular receptors, growth factors, cytokines or sections thereof.
  • the expression cassette is introduced into the cells without additional bacterial nucleic acid sequence sections.
  • the expression cassette is contained in a nucleic acid construct comprising only eukaryotic sequence sections, e.g.
  • nucleic acid sequence sections comprising or consisting of the expression cassette, optionally a eukaryotic selection marker, nucleic acid sequence sections having homology to genomic nucleic acid sequence sections of the cells line for allowing homologous recombination, and/or fused remainders of recombinase recognition sites flanking the expression cassette, e.g. introduced by transient or permanent transfection.
  • the cells can be contacted with attenuated virus, e.g. for producing a vaccine comprising the viral particles in a pharmaceutically acceptable formulation.
  • attenuated virus e.g. for producing a vaccine comprising the viral particles in a pharmaceutically acceptable formulation.
  • examples for viral particles are the modified Ankara virus strain of the vaccinia virus, attenuated strains of virus having specificity for humans and/or animals.
  • the process for producing protein comprises the steps of
  • the process preferably comprises the steps of
  • the viral particle can be an attenuated virus, a wild-type virus, or comprise a synthetic nucleic acid sequence in viral coat protein.
  • Example 1 Generation of an avian cell line
  • a cell line of the invention was generated from avian cells obtained from embryonic tissue of an Anas platyrhinchos embryo at day 13, cultivated in MEM, supplemented with 10% FBS for 45 - 50 passages, each passage comprising growth under cell culture conditions in static cell culture flasks to 70 - 100% confluence. It was observed that at passage 40, cell proliferation was significantly decreased. Passaging was continued until passage 144. In further passaging, e.g. in the subsequent 8 passages, the concentration of serum was reduced, in one step or, as preferred in steps of 0.5 to 1%, to a total of 2% FBS in cell culture medium, preferably in AdMEM, with subsequent further passaging in medium with a content of 1% FBS.
  • serum-free medium preferably having a composition of serum-free medium EBx GRO-I without glutamine, suitable for embryonic stem cells as available from SAFC Biosciences, Andover (UK) under catalogue No. 14530C, preferably with glutamine added according to the manufacturer's instructions.
  • serum-free medium it was observed that cells started to grow in free suspension. The cells could be transferred to a bioreactor for suspended growth, e.g. with agitation of the serum-free medium, e.g. by orbital shaking.
  • One cell line generated by this process was deposited under accession number DSM ACC3149.
  • the cell line growing in serum-free medium in suspension could be used for expression of heterologous soluble protein or, using infection by a viral particle, for production of attenuated virus.
  • the growth was monitored by measuring the doubling time. Susceptibility to infection was measured by determination of the infectious titer of an aliquot of the adherent cells using a virus, e.g. Influenza A Puerto Rico 8/34/ (Mount Sinai strain), using the plaque assay on an MDCK cell layer.
  • a virus e.g. Influenza A Puerto Rico 8/34/ (Mount Sinai strain)
  • the plaque assay on an MDCK cell layer.
  • the hemagglutination reaction can be used, which is also applicable for non-infectious virus particles.
  • Neuraminidase activity was measured in cells growing in suspension by the NA-STAR assay (available from Applied Biosystems, USA), and the heterologous protein expression was measured using a reporter gene assay by transfecting the cells with a nucleic acid construct containing an expression cassette encoding a reporter gene, e.g. G-Luc (Gaussia Luciferase), C-Luc (Cypridina Luciferase) as a reporter gene by measuring luciferase activities (e.g. using the GLuc or C-Luc reporter gene assay available from NEN BioLabs).
  • G-Luc Gaussia Luciferase
  • C-Luc Cypridina Luciferase
  • the doubling or generation times were determined to 22h at passage 7 and to 28.8h at passage 30.
  • the population doubling time at passage 146 was determined to 20.1h in AdMEM, 1% FBS, conditioned by adding the same volume of 1d culture supernatant obtained from a confluent culture of EFB1 (duck fibroblast) cells in the same medium, and to 28.7h in AdMEM, 1% FBS without this conditioning.
  • an infectious titer of 11-17 pfu/cell and day was determined at passages 58-66 for the cell line cultivated in 1% FBS AdMEM medium.
  • cells were passaged for at least 40 passages in ExCell EBxGro-I medium supplemented to a final concentration of 2.5 mM glutamine.
  • human influenza virus A/Puerto Rico/8/1934 H1N1 (Influenza A PR8) was produced as an example for a viral particle. Briefly, cells were grown in static culture in 1% FBS MEM medium to confluence and infected with a multiplicity of infection (MOI) of 0.2. Following further incubation for 24h under cell culture conditions. The titer of viral particles was determined from culture supernatant by the plaque assay on MDCK cells.
  • Figure 3 shows the cell growth of SEFB1 of the invention infected with Influenza A virus PR8 strain at an MOI of 0.01 at day 6 of cultivation in suspension , as indicated by the arrow for addition of the infectious viral particles.
  • Cells were cultivated in 10 mL growth medium (EBx GroI) in a bioreactor 50 (TPP, 50ml on an orbital shaker (1 cm orbit, 220 rpm). The lytic effect of the virus is evident.
  • Figure 4 shows the relative productivity of the cells of the invention for Influenza A PR8 in the neuraminidase assay (NA_STAR) with luminescence detection in comparison to virus production in adherently growing (adh) MDCK cells (100%).
  • NA neuraminidase activity
  • NASTAR a luminometric assay
  • Parallel experiments (Exp) 1 to 4 for the cell line of the invention show that 21-53% of the productivity in MDCK cells was reached in these initial experiments.
  • EX-CELL EBx GRO-I medium was used, and EX-CELL EBx PRO-I medium was used in experiment 4.
  • Example 3 Production of soluble secretory protein in suspension culture
  • an expression cassette encoding secretory Cypridina luciferase (C-Luc) under the control of the SV40 promoter was transiently introduced into cells generated according to Example 1 and into HEK 293s suspension cells for comparison.
  • the expression cassette was contained in a bacterial shuttle plasmid. Cells were transfected by contacting cells with the DNA containing the expression cassette in vector pSV-CLuc in the presence of polyethylene imine (PEI).
  • PEI polyethylene imine
  • Figure 5 shows the activity of secreted C-Luc in cell-free medium of the comparative 293s cell culture under the control of the SV40e (SV40 early) promoter.
  • Cells were cultivated in 10 mL growth medium (SFM II) in a bioreactor 50 (TPP) on an orbital shaker (1 cm orbit, 220 rpm).
  • Cells were transfected with the plasmid containing an expression cassette comprising SV40e C-Luc using a 3:1 ratio of PEI:DNA.
  • Cells were cultivated for the indicated duration, Cypridina luciferase (C-Luc) production was determined daily from supernatant after 10-fold dilution in a luminometric assay. Light units are indicated as l.u.
  • Figure 6 shows the activity of secreted C-Luc in cell-free medium of a cell line of the invention, sEFB1.
  • Cells were cultivated in 10 mL growth medium (EBx Pro-I) in a bioreactor (BioReactor 50, TPP) with orbital shaking (1 cm orbit, 220 rpm).
  • Cells were transfected with the plasmid containing an expression cassette comprising SV40e C-Luc using a 3:1 ratio of PEI:DNA.
  • Cells were cultivated for the indicated duration, Cypridina luciferase (C-Luc) production was determined daily from supernatant after 10-fold dilution in a luminometric assay.
  • ⁇ and ⁇ indicate results from individual experiments using pSV-CLuc. Measurement results show that soluble secretory protein was produced to a 3-fold higher concentration in the cell line of the invention in comparison to suspended HEK 293s cells.
  • Example 4 Production of soluble non-secretory protein in suspension culture
  • an expression cassette encoding firefly luciferase (F-Luc) under the control of the human cytomegalovirus promoter (hCMV) was constructed on a minicircle, i.e. on a plasmid devoid of sections of bacterial origin and transiently transfected into comparative HEK 293s cells and in cells generated according to Example 1, respectively, using PEI for transfection. Briefly, the minicircle did not contain nucleic acid sections used for replication in bacteria and was produced by internal recombination using Flp recombinase on the two Flp-specific FRT recognition sites flanking the expression cassette.
  • the minicircle essentially consisted of the expression cassette containing the coding sequence for the protein (F-Luc) between an upstream promoter element and a downstream polyadenylation site and the joined remainders of the FRT recombinase recognition sites.
  • Luciferase activities were measured for 4 days from cells isolated from the culture, and cell number was determined for calculation of relative activities. Productivity values are normalized to cell number and daily production. It was found that non-secretory protein can be produced in the cells of the invention in suspended culture.
  • minicircles perform equally well in 293s cells and in sEFB1 cells of the invention.
  • sEFB1 cells the luciferase expression was approx. threefold higher from minicircles (black bar) compared to the original plasmids (white bar) containing sequence sections of bacterial origin (ori, selection marker ).
  • sequence sections of bacterial origin ori, selection marker .
  • the higher expression from minicircle compared to plasmids was also found in HEK 293s cells.
  • Example 5 Production of two soluble proteins from coupled expression cassettes in suspension culture
  • two expression cassettes were coupled, a first expression cassette containing a promoter upstream a first protein coding sequence, and in 3' an adjacent second expression cassette containing an IRES element as a promoter element upstream a second protein coding sequence, with a polyadenylation (pA) element downstream of the second protein coding sequence.
  • the first protein coding sequence was secreted alkaline phosphatase (SEAP), which due to being secretory was analysed from the cell-free culture medium.
  • a splice donor site and a splice acceptor site were arranged between the promoter and the first coding sequence.
  • the second coding sequence encoded the non-secretory F-Luc.
  • the genetic transcriptional control elements were chosen from the human cytomegalovirus immediate early (IE) promoter/enhancer (hCMV), the modulator and unique region (MUR) upstream of the human cytomegalovirus IE promoter combined with the human cytomegalovirus immediate early (IE) promoter/enhancer (hCMV) (MUR CMV), the Simian virus 40 early promoter (SV40e), the long terminal repeat region (LTR) of the myeloproliferative sarcoma virus harbouring the retroviral promoter (MPSV), the Rous sarcoma virus promoter (RSV).
  • IE cytomegalovirus immediate early
  • MUR CMV modulator and unique region
  • SV40e Simian virus 40 early promoter
  • LTR long terminal repeat region
  • MPSV myeloproliferative sarcoma virus harbouring the retroviral promoter
  • RSV Rous sarcoma virus promoter
  • the translational control elements (internal ribosome entry sites, IRES) were chosen from the poliovirus 5'-untranslated region (Polio-5'-UTR), the bovine viral diarrhea virus 5'-UTR (BVDV-5'-UTR), and the glucose-6-phosphate isomerase 5'-UTR (G6PI-5'-UTR).
  • the coding sequence was SEAP, the secreted alkaline phosphatase gene, and F-Luc, the firefly luciferase gene, respectively.
  • the splice donor (SD) and splice acceptor (SA) regions were from SV40e, the polyadenylation site (pA) originates from the SV40 early pA.
  • Figure 8 schematically shows the arrangement of the genetic elements.
  • Figures 9 and 10 show data for protein expression by way of activities of the proteins expressed. In each set of columns, the left column indicates the activity 1 day after transfection, the centre column indicates the activity after 2 days, and the right column indicates the activity after 3 days.
  • Cells were transfected with plasmids containing one of the monocistronic and bicistronic expression cassettes.
  • the SV40 early promoter was contained in pNSV Luc and pSBC cassettes ( Dirks et al.
  • the cell lines of the invention are suitable for simultaneous production of at least two proteins, at least one of which can be a secretory and/or a non-secretory protein, which can be encoded by adjacent expression cassettes on one common nucleic acid construct, e.g. on an at least bicistronic nucleic acid construct. Further, the results show that the expression level of each protein is dependent on the promoter element functionally coupled to the coding sequence, and that accordingly, the relative expression of different proteins produced in a cell line can be controlled by the promoter element functionally coupled to the coding sequence.
  • the promoter strength in the cell line i.e. the relative level of expression from the coding sequence functionally coupled to the region having promoter activity, is MUR CMV > hCMV ⁇ SV ⁇ 40 ⁇ e > RSV LTR > > MPSV LTR and for the elements suitable for internal initiation of translation of mRNA, IRES, the strength of initiation of translation is BVDV >> G6PI - poliovirus.
EP20120166489 2012-05-02 2012-05-02 Lignée cellulaire aviaire et son utilisation dans la production de protéines Withdrawn EP2660316A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
EP20120166489 EP2660316A1 (fr) 2012-05-02 2012-05-02 Lignée cellulaire aviaire et son utilisation dans la production de protéines
PCT/EP2013/059117 WO2013164389A1 (fr) 2012-05-02 2013-05-02 Lignée cellulaire aviaire et son utilisation dans la production de protéine
US14/396,336 US20150104830A1 (en) 2012-05-02 2013-05-02 Avian cell line and its use in production of protein
EP13723038.9A EP2844740A1 (fr) 2012-05-02 2013-05-02 Lignée cellulaire aviaire et son utilisation dans la production de protéine

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WO2005007840A1 (fr) 2003-07-22 2005-01-27 Vivalis Production de poxvirus avec des lignees cellulaires aviaires adherentes ou non adherentes
WO2005042728A2 (fr) 2003-11-03 2005-05-12 Probiogen Ag Lignees de cellules aviaires immortalisees pour la production de virus
EP1483369B1 (fr) 2002-03-08 2008-09-10 Vivalis Lignées de cellules aviaires utiles pour la production de substances d'intérêt
EP1985305A1 (fr) 2007-04-24 2008-10-29 Vivalis Lignées de cellules souches dérivées d'embryons de canard pour la fabrication de vaccins viraux

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EP1483369B1 (fr) 2002-03-08 2008-09-10 Vivalis Lignées de cellules aviaires utiles pour la production de substances d'intérêt
US20110294209A1 (en) * 2002-03-08 2011-12-01 Vivalis Methods for producing nonadherent avian cell lines
WO2005007840A1 (fr) 2003-07-22 2005-01-27 Vivalis Production de poxvirus avec des lignees cellulaires aviaires adherentes ou non adherentes
EP1646715B1 (fr) 2003-07-22 2010-05-12 Vivalis Production de virus de la vaccine en utilisant des lignées de cellules aviaires adhérentes ou non adhérentes
WO2005042728A2 (fr) 2003-11-03 2005-05-12 Probiogen Ag Lignees de cellules aviaires immortalisees pour la production de virus
EP1985305A1 (fr) 2007-04-24 2008-10-29 Vivalis Lignées de cellules souches dérivées d'embryons de canard pour la fabrication de vaccins viraux

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